The first evidence for the importance of heavy chain phosphorylation of AMII was our data in the early 1980s showing that its actin-activated MgATPase activity was inhibited by phosphorylation of 1 to 3 serine residues in the 29-residue non-helical tailpiece of each of the two heavy chains. For a period of about 10 years from the mid-1980s to mid-1990s, we had investigated the possible mechanism of this inhibition. The one consistent result was that heteropolymers of unphosphorylated (active) myosin and phosphorylated (inactive) myosin had significantly less actin-activated ATPase activity than equivalent mixtures of homopolymers, i.e. regulation of AMII seemed to be at the level of filaments with phosphorylated AMII inactivating unphosphorylated AMII when both are in the same filaments. Interestingly, the phosphorylation sites at the C-terminal end of the tail are 80-nm distant from the ATPase site in the globular separated by a coiled-coil helix. At that time, neither we nor others had any evidence that phosphorylation of the non-helical tailpiece affected the filament structure. We have reinvestigated this problem by studying the filament structure and actin-activated ATPase activity of recombinant wild-type and mutant AMIIs. We now find, by quantitative mass spectrometry, that incubation with myosin II heavy chain kinase phosphorylates four serines in the non-helical tailpiece (NHT) of full-length AMII, and Ser639 in loop 2 of the motor domain of full-length AMII, heavy meromyosin (HMM) and sub-fragment 1 (S1). The actin-activated ATPase of all of the myosin constructs is inhibited by phosphorylaion and, since HMM and S1 lack the myosin tail, inhibition of actin-activaed ATPase activity must result from phosphorylation of Ser639. Consistent with this conclusion, the S639A mutant of full-length AMII is not inhibited by phosphorylation of the NHT-serines or their mutation to glutamate. Conversely, S639D mutants of both S1 and full-length AMII are enzymatically inactive irrespective of the phosphorylation state of the NHT-serines of full-length AMII. Kinetic analysis of each step in the myosin and actomyosin ATPase cycles of recombinant wild-type and S639D S1 showed that the release of Pi from the actomyosin ADP-Pi complex is the step predominantly inhibited by the S639D mutation. Although phosphorylation of the serines in the non-helical tailpiece does not affect enzymatic activity, we find it does affect the structure of the AMII minifilaments. As seen by electron microscopy of rotary shadowed filaments, bipolar tetrameric, hexameric and octameric minifilaments of full-length AMII with their NHT-serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than non-phosphorylated, non-mutated AMII irrespective of the phosphorylation state of Ser639. Thus, we have shown tha the actin-activated ATPase activity of AMII is regulated by phosphorylation of Ser639 in the motor domain and the filament structure of AMII is modified by phosphorylation of one or more of the four serines in the NHT. These results have significant implications for the regulation of the filament structure of mammalian non-muscle myosins IIs whose NHT-serines and threonines can also be phosphorylated both in vitro and in vivo.